PROJECT DESCRIPTION The nuclear envelope is a subdomain of the endoplasmic reticulum (ER) that surrounds and protects the genome. Contrary to prior dogma, the nuclear envelope is not a stable structure, but is highly dynamic in both interphase and mitosis. Defects in nuclear envelope dynamics are common to cancer cells and to genetic disorders caused by mutations in nuclear envelope proteins that include muscular dystrophy and progeria. Thus, elucidating mechanisms that underlie nuclear envelope dynamics will inform on our understanding of disease mechanisms ranging from cancer to accelerated aging. An underlying principle that drives membrane dynamics at nearly all membrane-bound organelles is the composition of bilayer phospholipids, yet little is known about the role of phospholipids in nuclear envelope dynamics. This proposal focuses on a distinct regulator of phospholipid synthesis, CTDNEP1 (formerly known as Dullard), that is enriched at the nuclear envelope and is required for nuclear envelope dynamics. CTDNEP1 and its obligate binding partner NEP1R1 are a ubiquitously conserved protein phosphatase complex that activate the phosphatidic acid phosphatase lipin; lipin's activity is central to the de novo phospholipid synthesis. The work proposed here has two main aims: 1) to establish the functional significance of CTDNEP1-NEP1R1 localization at the nuclear envelope in local regulation of phospholipid synthesis and nuclear envelope dynamics and 2) to elucidate a role for nuclear envelope lipids in protecting genome integrity by regulating mitotic nuclear envelope reformation. Completion of these goals will elucidate a potentially significant, yet understudied role for lipid-mediated regulation of membrane dynamics at the nuclear envelope to ensure genome protection. Aim 1 will focus on the role of a conserved region in NEP1R1 that we discovered targets/retains CTDNEP1 in the nuclear envelope/ER in human cells. We will identify mutants in NEP1R1 that specifically disrupt enrichment of CTDNEP1-NEP1R1 at the nuclear envelope so that it is a more generic ER protein and assess the effects on lipid composition and nuclear envelope dynamics. Aim 2 builds on our preliminary data that CTDNEP1 regulates nuclear envelope reformation and prevents micronucleus formation in dividing cancer cells. These data suggest that core nuclear envelope proteins may be regulated by specific phospholipids to form the nuclear envelope from ER-derived membranes. We will use endogenous tags combined with conventional and super-resolution live-cell imaging to systematically define the localization and dynamics of core nuclear envelope proteins relative to nascent nuclear membranes during nuclear envelope reformation in wild type and ctdnep1-deficient cancer cells. In vitro lipid binding assays will identify domains in nuclear envelope proteins that directly bind to major phospholipids. Such studies will allow the design of mutant proteins with altered lipid binding properties to test for functional significance in ER membrane recruitment and remodeling to generate the nuclear envelope.